Use of scatter factor to enhance angiogenesis

ABSTRACT

This invention relates to a method of enhancing wound healing utilizing scatter factor, either alone or in combination with a growth factor.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under NIH Grant No.CA50516. As such, the government has certain rights in the invention.

This application is a continuation of application Ser. No. 08/138,667filed on Oct. 18, 1993 now abandoned.

FIELD OF THE INVENTION

This invention relates to a method of enhancing wound healing and to amethod of enhancing organ transplantation comprising the administrationof scatter factor to promote angiogenesis.

BACKGROUND OF THE INVENTION

Scatter factor has previously been described as a cytokine which issecreted by fibroblasts (see Stoker et al., J. Cell Sci., Vol. 77, pp.209-223 (1985) and Stoker et al., Nature (London), Vol. 327, pp. 238-242(1987)) and by vascular smooth muscle cells (see Rosen et al., In VitroCell Dev. Biol., Vol. 25, pp. 163-173 (1989)). Scatter factor has beenshown to disperse cohesive epithelial colonies and stimulate cellmotility. In addition, scatter factor has been shown to be identical tohepatocyte growth factor (HGF) (see Weidner et al., Proc. Nat'l. Acad.Sci. USA, Vol. 88, pp. 7001-7005 (1991) and Bhargava et al., Cell GrowthDiffer., Vol. 3, pp. 11-20 (1992)), which is an independentlycharacterized serum mitogen (see Miyazawa et al., Biochem. Biophys. Res.Commun., Vol. 169, pp. 967-973 (1989) and Nakamura et al., Nature(London), Vol. 342, pp. 440-443 (1989)). Scatter factor induces kidneyepithelial cells in a collagen matrix to form branching networks oftubules, suggesting that it can also act as a morphogen (see Montesanoet al., Cell, Vol. 67, pp. 901-908 (1991)).

Scatter factor (HGF) is a basic heparin-binding glycoprotein consistingof a heavy (58 kDa) and a light (31 kDa) subunit. It has 38% amino acidsequence identity with the proenzyme plasminogen (see Nakamura et al.,Nature (London), Vol. 342, pp. 440-443 (1989)) and is thus related tothe blood coagulation family of proteases. Its receptor in epitheliumhas been identified as the c-met protooncogene product, a transmembranetyrosine kinase (see Bottaro et al., Science, Vol. 251, pp. 802-804(1991) and Naldini et al., Oncogene, Vol. 6, pp. 501-504 (1991)).

Scatter factor has been found to stimulate endothelial chemotactic andrandom migration in Boyden chambers (see Rosen et al., Proc. Soc. Exp.Biol. Med., Vol. 195, pp. 34-43 (1990)); migration from carrier beads toflat surfaces (see Rosen et al., Proc. Soc. Exp. Biol. Med., Vol. 195,pp. 34-43 (1990)); formation of capillary-like tubes (see Rosen et al.,Cell Motility Factors, (Birkhauser, Basel) pp. 76-88 (1991)) and DNAsynthesis (see Rubin et al., Proc. Nat'l. Acad. Sci. USA, Vol. 88, pp.415-419 (1991)). In addition, preliminary studies have suggested thatscatter factor induces endothelial secretion of plasminogen activators(see Rosen et al., Cell Motility Factors, (Birkhauser, Basel) pp. 76-88(1991)).

The term "angiogenesis", as used herein, refers to the formation ofblood vessels. Specifically, angiogenesis is a multistep process inwhich endothelial cells focally degrade and invade through their ownbasement membrane, migrate through interstitial stroma toward anangiogenic stimulus, proliferate proximal to the migrating tip, organizeinto blood vessels, and reattach to newly synthesized basement membrane(see Folkman et al., Adv. Cancer Res., Vol. 43, pp. 175-203 (1985)).These processes are controlled by soluble factors and by theextracellular matrix (see Ingber et al., Cell, Vol. 58, pp. 803-805(1985)).

Because proteases, such as plasminogen activators (the endothelialsecretion of which is induced by scatter factor) are required during theearly stages of angiogenesis, and since endothelial cell migration,proliferation and capillary tube formation occur during angiogenesis,the inventors hypothesized that scatter factor might enhance angiogenicactivity in vivo. In addition, it is desirable to enhance angiogenicactivity so that wound healing and organ transplantation can beenhanced.

It is therefore an object of this invention to provide a method ofenhancing angiogenic activity.

It is a further object of this invention to provide a method ofenhancing wound healing.

It is a still further object of this invention to provide a method ofenhancing organ transplantation.

SUMMARY OF THE INVENTION

This invention is directed to a method of promoting angiogenesiscomprising the administration of scatter factor. Scatter factor isadministered topically, intravenously, intramuscularly, intradermally,subcutaneously or intraperitoneally, and is administered either alone orin combination with a growth factor. Scatter factor can be administeredto promote angiogenesis so as to enhance wound healing and organtransplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description, as well as further objects and features ofthe present invention, will be more fully understood by reference to thefollowing detailed description of the presently preferred, albeitillustrative, embodiments of the present invention when taken inconjunction with the accompanying drawing wherein:

FIG. 1 is comprised of FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D. FIG. 1represents the result of a murine angiogenesis assay. FIG. 1A showsMatrigel plugs (arrowheads) before excision of the plugs. FIG. 1B showsplugs (arrowheads) after excision of the plugs. FIG. 1C shows thequantification by digital image analysis for athymic mice. FIG. 1D showsthe quantitation by digital image analysis for C57BL mice;

FIG. 2 is comprised of FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. FIG. 2represents the microscopic appearance of Matrigel plugs. FIG. 2Arepresents sections of plugs from athymic control (0 ng scatter factor)mice. FIG. 2B represents sections of plugs from athymic mice whichcontain 2 ng scatter factor. FIG. 2C represents sections of plugs fromathymic mice which contain 20 ng scatter factor. FIG. 2D representssections of plugs from athymic mice which contain 200 ng of scatterfactor;

FIG. 3 is comprised of FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D and FIG. 3E.FIG. 3 represents scatter factor-induced angiogenesis in rat corneas. Asshown in FIG. 3A, no angiogenic response was observed in control pelletscontaining PBS. As shown in FIG. 3B, the response for 50 ng scatterfactor was positive but weak in comparison with high concentrations ofscatter factor. As shown in FIG. 3C, and FIG. 3D, scatter factorconcentrations of 100 ng and 500 ng, respectively, gave strong positiveresponses. FIG. 3E shows a strong angiogenic response which was inducedby 150 ng of basic FGF, a positive control;

FIG. 4 is comprised of FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E.FIG. 4 represents stimulation of plasminogen activator expression byscatter factor. FIG. 4A shows secreted activity during a 6 hourcollection interval. FIG. 4B shows secreted activity intracellularly.FIG. 4C shows total (secreted plus intracellular) activity. FIG. 4D andFIG. 4E show plasminogen activator activity in medium and in lysatesfrom cells treated with scatter factor at 20 ng/ml assayed in thepresence of goat anti-human urokinase IgG (αuPA), goat anti-human tissuetype PA IgG (αtPA) or goat nonimmune IgG (NI IgG) (200 μg/ml),respectively; and

FIG. 5 is comprised of FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D. FIG. 5represents the results of immunohistochemical staining of skin biopsysamples for scatter factor. FIG. 5A, FIG. 5B and FIG. 5C showimmunohistochemical staining of psoriatic plagues. FIG. 5D showsimmunohistochemical staining of normal skin from a patient withpsoriasis.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to a method of promoting angiogenesis byadministering scatter factor. Scatter factor can be administeredtopically, intravenously, intramuscularly, intradermally, subcutaneouslyor intraperitoneally. The amount of scatter factor to be administered isabout 0.1-1000 ng/kg body weight.

Angiogenesis can also be enhanced by administering scatter factor incombination with a growth factor. The growth factor can be selected fromthe group consisting of TGF-α, FGF and PDGF.

The inventors have discovered that angiogenesis is promoted by theadministration of scatter factor. Hence, scatter factor can be used toenhance wound healing and organ transplantation, including thetransplantation of artificial organs. This invention is thereforedirected to a method of enhancing wound healing and to a method ofenhancing organ transplantation. In addition, scatter factor can be usedto accelerate endothelial cell coverage of vascular grafts in order toprevent graft failure due to reocclusion, and to enhance skin grafting.Further, antibodies to scatter factor can be used to treat tumors and toprevent tumor growth.

In order to prepare scatter factor preparations, mouse scatter factorwas purified from serum-free culture medium from ras-transformedNIH/23T3 cells (clone D4) by cation-exchange chromatography as describedby Rosen et al., Proc. Soc. Exp. Biol. Med., Vol. 195, pp. 34-43 (1990),followed by immunoaffinity chromatography and ultrafiltration.Recombinant human HGF (rhHGF) was provided by Toshikazu Nakamura (KyushuUniversity, Fukuoka, Japan). Scatter factor (HGF) is commerciallyavailable from Collaborative Research, Bedford, Mass.

In order to make the antibody preparations, antisera to native humanplacental scatter factor and rhHGF were prepared by immunizing rabbitswith purified factors (see Bhargava et al., Cell Growth Differ., Vol. 3,pp. 11-20 (1992) and Bhargava et al., Cell Motility Factors,(Birkhauser, Basel) pp. 63-75 (1991)). A chicken egg yolk antibody tohuman placental scatter factor was prepared by immunizing two WhiteLeghorn hens, 22-24 weeks old, with 500 μg of human placental scatterfactor emulsified in complete Freund's adjuvant. Booster injections weregiven 14 to 28 days later, and the eggs were collected daily. The IgGfraction from seven eggs was extracted and partially purified by themethods described by Polson et al., Immunol. Commun., Vol. 9, pp.495-514 (1980). The final preparation contained 80 μg of protein per mlin phosphate-buffered saline (PBS). Antibody specificity was establishedby recognition of mouse and human scatter factors on immunoblots,specific binding of scatter factor to antibody-Sepharose columns, andinhibition of the in vitro biologic activities of mouse and humanscatter factor.

In order to perform plasminogen activator assays, bovine brainmicrovessel endothelial cells (BBEC) were isolated from brain cortexafter removal of the pia mater, identified as endothelial, and culturedby standard techniques. BBEC (passage 10-12) at about 80% confluency in60 mm Petri dishes were treated with mouse scatter factor for 24 hours,washed, and incubated for 6 hours in 2.5 ml of serum-free Dulbrecco'smodified Eagle's medium (DMEM) to collect secreted proteins. The cellswere washed, scraped into PBS, collected in 0.5 ml of PBS bycentrifugation, and lysed by sonication. Aliquots of medium and celllysates were assayed for PA activity by a two-step chromogenic reactionas described by Coleman et al., Ann. N.Y. Acad. Sci., Vol. 370, pp.617-626 (1991). Human high molecular weight urokinase (AmericanDiagnostica, Greenwich, Conn.) was used as the standard. The proteincontent of the lysate was determined by using the Bradford dye-bindingassay (Bio-Rad).

In order to perform the murine angiogenesis assay, angiogenesis wasassayed as growth of blood vessels from subcutaneous tissue into a solidgel of basement membrane containing the test sample. Matrigel (7 mg in0.5 ml; Collaborative Research) in liquid form at 4° C. was mixed withscatter factor and injected into the abdominal subcutaneous tissues ofathymic XID nude beige mice or C57BL/6 mice. Matrigel rapidly forms asolid gel at body temperature, trapping the factor to allow slow releaseand prolonged exposure to surrounding tissues. After 10 days, the micewere sacrificed and the Matrigel plugs were excised and fixed in 4%formaldehyde in phosphate buffer. Plugs were embedded in paraffin,sectioned, stained with Masson's trichrome (which stains endothelialcells reddish-purple and stains the Matrigel violet or pale green), andexamined for ingrowth of blood vessels. Vessel formation was quantitatedfrom stained sections using the Optimax digital image analyzer connectedto an Olympus microscope (see Grant et al., Cell, Vol. 58, pp. 933-943(1989)). Results were expressed as mean vessel area per field±SEM(arbitrary units) or as total vessel area (mm²) in 20 random fields.

In order to perform the rat cornea angiogenesis assay, angiogenesis wasassayed in the avascular rate cornea, as described by Polverini et al.,Lab. Invest., vol. 51, pp. 635-642 (1984). Test samples were combined1:1 with a sterile solution of Hydron (Interferon Laboratories, NewBrunswick, N.J.) and air-dried overnight. A 5 μl pellet was insertedinto a surgically created pocket in the corneal stroma and positioned1-1.5 mm from the limbus. Corneas were examined daily with a dissectingmicroscope for up to 7 days for capillary growth. Assay responses werescored as positive if sustained directional ingrowth of capillarysprouts and hairpin loops occurred during the observation period.Responses were scored as negative either when no neovascularization wasdetected or when only an occasional sprout or hairpin loop was observedthat showed no evidence of sustained directional ingrowth. After 7 days,corneas were perfused with colloidal carbon, and whole-mountpreparations were examined and photographed.

To study immunohistochemistry, five-micometer-thick cryostat sectionswere prepared from biopsy samples of plaques or of areas of normal skinin patients with active psoriasis. The sections were stained by using anavidin-biotin immunoperoxidase technique (see Griffiths et al., Am.Acad. Dermatol., Vol. 20, pp. 617-629 (1989)). The chromogen was Texasred conjugated to avidin. The primary antibody was rabbit polyclonalantiserum to purified native human placental scatter factor or to rhHGF(1:1000 dilution). Nonimmune rabbit serum (1:1000) was used as anegative control.

Two different in vivo assays were used to evaluate the angiogenicactivity of mouse scatter factor. In the first assay, the murineangiogenesis assay, samples mixed with Matrigel, a matrix ofreconstituted basement membrane, were injected subcutaneously into mice.After 10 days, the mice were sacrificed for histologic and morphometricanalysis of Matrigel plugs. Control plugs were found to be pale pink,while plugs containing scatter factor were found to be bright red andoften contained superficial blood vessels (see FIG. 1A and FIG. 1B).

Histologic analysis showed little cellularity in control plugs (see FIG.2A). Plugs containing 2 ng of scatter factor often had increased numbersof cells (see FIG. 2B), 90% of which stained for factor VIII antigen, anendothelial cell marker (not shown). At 20 ng of scatter factor, cellnumber was increased, and vessels were present (FIG. 2C). At 200 ng ofscatter factor, plugs were even more cellular, with endothelial cellsmaking up 50-60% of the cell population. Many large vessels containingsmooth muscle cells were seen (see FIG. 2D).

Morphometric analysis of vessel area (see Grant et al., Cell, Vol. 58,pp. 933-943 (1989)) revealed a dose-dependent angiogenic response inathymic (FIG. 1C) and C57BL (FIG. 1D) mice, with half-maximal andmaximal responses at about 20 and 200 ng, respectively. Histologicexamination at day 10 showed no evidence of inflammation in scatterfactor-containing plugs in athymic mice. In C57BL, no inflammation wasobserved at ≦200 ng of scatter factor, but leukocytic infiltration waspresent in tissue surrounding the plugs at ≦2000 ng of scatter factor.

In the second assay, samples were implanted in the avascular rat corneato allow ingrowth of blood vessels from the limbus. Control implantsgave no positive responses (see Table 1, below, and FIG. 3A), whileimplants containing mouse scatter factor induced a dose-dependentcorneal neovascularization. Responses at 50 ng (FIG. 3B) were reduced inintensity compared with those at 100 and 500 ng (FIG. 3C and FIG. 3D,respectively). The maximal response to scatter factor was observed atdoses of ≦100 ng and was similar to the response to 150 ng of humanbasic FGF, a positive control (see FIG. 3E).

                  TABLE 1                                                         ______________________________________                                        Neovascular responses induced                                                 in rat corneas by scatter factor (SF)                                                         Corneal                                                                       neovascularization                                                              Positive                                                    Content of pellet responses                                                                              %                                                  ______________________________________                                        Negative controls                                                             Sham implant      0/3      0                                                  Hydron            0/2      0                                                  PBS               0/2      0                                                  Positive control                                                              Basic FGF (150 ng)                                                                              4/4      100                                                scatter factor (SF)                                                             5 ng            0.4      0                                                   50 ng             3/5*    60                                                  100 ng           5/5      100                                                 500 ng           5/5      100                                                1000 ng             5/5+   100                                                ______________________________________                                         *Responses were much weaker in intensity compared with implants containin     100 or 500 ng of scatter factor.                                              +Corneas showed significant inflammation.                                

rhHGF also induced angiogenesis in the rat cornea (see Table 2, below).At 100 ng, positive responses were observed in four of five implants. At500 ng of rhHGF, all five implants gave positive responses. Chicken andrabbit antibodies to human placental scatter factor strongly inhibitedthe angiogenic responses to mouse scatter factor and rhHGF, but not tobasic FGF (see Table 2).

                  TABLE 2                                                         ______________________________________                                        Neovascular responses induced in rat corneas                                  by native mouse scatter factor (SF)                                           and rhHGF with or without antibody (Ab)                                                          Corneal                                                                       neovascularization                                                              Positive                                                 Content of pellet    responses                                                                              %                                               ______________________________________                                        Controls                                                                      Hydron + PBS         0/8      0                                               Chicken Ab           0/4      0                                               Rabbit Ab (Ab 978)   0/3      0                                               Basic FGF (150 ng)   3/3      100                                             Basic FGF (150 ng) + rabbit Ab                                                                     3/3      100                                             Factor ± Ab                                                                Mouse SF (100 ng)    3/3      100                                             Mouse SF (100 ng) + chicken Ab                                                                      1/5*    20                                              rhHGF (100 ng)       4/5      80                                              rhHGF (500 ng)         5/5+   100                                             rhHGF (100 ng) + chicken Ab                                                                         2/5*    33                                              rh HGF (100 ng) + rabbit Ab                                                                        0/5      0                                               ______________________________________                                         Antibodies were diluted in PBS. Final dilutions after mixing with Hydron      were 1:20 for the chicken antibodies and 1:200 for the rabbit antibodies.     *Responses scored as positive were very weak.                                 +This concentration of rhHGF was inflammatory.                           

To assess inflammation, corneas were examined by direct stereomicroscopydaily for the duration of the experiments. Corneas chosen at random wereexamined histologically at 6, 12 and 24 hours and at 3, 5, and 7 daysafter implantation of scatter factor and control pellets. Inflammationwas not detected at lower angiogenic doses of scatter factor (50-500 ngof mouse scatter factor, 100 ng of rhHGF). At higher doses (50-500 ng ofmouse scatter factor, 500 ng of rhHGF), a prominent inflammatoryinfiltrate was observed. The majority of cells were monocytes andmacrophages, as judged by morphology and immunostaining for F4/80, amacrophage/monocyte marker.

Plasminogen activators convert plasminogen into plasmin, a potent serineprotease that lyses fibrin clots, degrades components of extracellularmatrix, and activates enzymes (e.g., procollagenases) that furtherdegrade matrix (see Saksela et al., Anu. Rev. Cell Biol., Vol. 4, pp.93-126 (1988)). The inventors have discovered that scatter factorinduces large dose-dependent increases in secreted (see FIG. 4A) andcell-associated (see FIG. 4B) plasminogen activator activity inmicrovascular endothelium (BBEC). Total plasminogen activator activity(secreted plus cell-associated) was increased 4-fold relative to controlwhen scatter factor was present at 20 ng/ml (≈0.2 nM). Similar resultswere. obtained in large vessel endothelium (not shown). Most of thesecreted and cell-associated plasminogen activator activity in BBEC wasblocked by antibodies to urokinase, but not by antibodies to tissueplasminogen activator (see FIG. 4D and FIG. 4E).

Angiogenesis is often associated with chronic inflammation diseases.Psoriasis is a common inflammatory skin disease characterized byprominent epidermal hyperplasia and neovascularization in the dermalpapillae. Frozen sections of biopsy samples from psoriatic plagues from10 patients each showed positive immunohistochemical staining forscatter factor in spindle-shaped and mononuclear cells within the dermalpapillae and papillary dermis. Antisera to human placental scatterfactor and rhHGF gave an identical staining pattern, as illustrated inFIG. 5A. Scatter factor-positive cells were arranged in a perivasculardistribution. Cells of the blood vessel wall did not stain for scatterfactor (see FIG. 5C). Normal skin from psoriasis patients or from normalsubjects showed little or no staining for scatter factor (FIG. 5D).Sections from psoriatic plaques treated with nonimmune serum as theprimary antibody (negative control) showed no staining (FIG. 5B).

Hence, the inventors have determined that physiologic quantities ofscatter factor 100-200 ng (≈1-2 pmol)! induced strong angiogenicresponses in two in vivo assays. It is likely that this angiogenicactivity is due, in part, to direct effects on endothelium since: (i)scatter factor stimulates endothelial migration, proliferation, and tubeformation in vitro; (ii) histologic studies showed no evidence ofinflammation at scatter factor doses that gave strong angiogenicresponses; and (iii) anti-scatter factor antibodies blocked theangiogenic responses. The inventors also found that scatter factorstimulates endothelial cell expression of urokinase. Urokinase, bound toits specific cells surface receptor, is thought to mediate focal,directed, extracellular proteolysis, which is required for endothelialcell invasion and migration during the early stages of angiogenesis.

Growth factors TGFβ, FGF, and platelet-derived growth factor (PDGF) arepresent in Matrigel and in the matrices of several tissues, includingthe cornea. The inventors have discovered that combinations of scatterfactor and either TGFβ, FGF, or PDGF provide greater stimulation ofendothelial tube formation in vitro than did the same agents usedindividually. The concentrations studied (1 ng/ml) were about 10 timesthose found in 250 μg of Matrigel, and scatter factor stronglystimulated tube formation on its own, by up to 8 times the amountstimulated by the control.

The major scatter factor producer cells are fibroblasts, smooth musclecells, and leukocytes. With rare exceptions, responder cells(epithelium, endothelium, melanocytes) are nonproducers. Theimmunohistochemical studies of psoriatic plaques suggest that scatterfactor is produced by cells located outside of the blood vessel wall.Studies by the inventors have indicated that cultured endothelial cellsexpress c-met mRNA and that immunoreactive c-met protein is present inblood vessel wall cells (endothelium and pericytes) in psoriaticplaques. This suggests that scatter factor may play a role inmicrovessel formation or elongation in psoriasis and that its likelymode of action is paracrine.

Scatter factor (HGF) stimulates motility, invasiveness, proliferation,and morphogenesis of epithelium, and it may be involved. in physiologicand pathologic processes such as embryogenesis, wound healing, organregeneration, inflammation, and tumor invasion. Angiogenesis is acomponent of each of these processes. Therefore, the in vivo biologicaction of scatter factor may be due, in part, to its effects on bothepithelial and vascular endothelial cells.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of various aspects of the invention. Thus, it isto be understood that numerous modifications may be made in theillustrative embodiments and other arrangements may be devised withoutdeparting from the spirit and scope of the invention.

We claim:
 1. A method for promoting angiogenesis in a mammal comprisingadministering to said mammal an amount of scatter factor effective topromote angiogenesis in said mammal.
 2. The method of claim 1 whereinthe mode of administration is selected from the group consisting oftopical, intravenous, intramuscular, intradermal, subcutaneous andintraperitoneal administration.
 3. The method of claim 1 wherein theamount of scatter factor administered is about 0.1-1000 ng/kg bodyweight of said mammal.
 4. The method of claim 1 which further comprisesthe step of simultaneously administering a growth factor.
 5. The methodof claim 4 wherein the growth factor is selected from the groupconsisting of TGF-β, FGF and PDGF.
 6. A method for enhancing woundhealing in a mammal comprising administering to said mammal an amount ofscatter factor effective to enhance wound healing in said mammal.
 7. Themethod of claim 6 wherein the mode of administration is selected fromthe group consisting of topical, intravenous, intramuscular,intradermal, subcutaneous and intraperitoneal administration.
 8. Themethod of claim 6 wherein the amount of scatter factor administered isabout 0.1-1000 ng/kg body weight of said mammal.
 9. The method of claim6 which further comprises the step of simultaneously administering agrowth factor.
 10. The method of claim 9 wherein the growth factor isselected from the group consisting of TGF-β, FGF and PDGF.